Abstract P5-02-16: SPEN is a biomarker for CDK4/6 inhibitor resistance in patients with metastatic hormone receptor positive (HR+)/HER2- breast cancer
Authors: Courtney T. van Geelen, Zhi Ling Teo, Peter Savas, Stephen J. Luen, Kylie A. Clarke, Sneha Sant, Karla J. Cowley, Franco Caramia, Kaylene J. Simpson, Fabrice Andre, Sarah-Jane Dawson, Richard Pearson, Sherene Loi
Journal: Cancer Research (2023)

Abstract Background: Despite significant improvements in the treatment of breast cancer (BC), metastatic disease remains the principal cause of BC-related death. Through analysis of rapid autopsy collected biospecimens, we have previously identified Split Ends (SPEN) alterations in patients with metastatic HR+/HER2- BC (Savas et al. 2016). The role of SPEN in BC is poorly defined. Here we aimed to further explore the function of this gene in metastatic HR+/HER2- metastatic BC (mBC). Methods: We explored the clinical and genomic characteristics of SPEN altered mBC in human sequencing datasets. We created a model of SPEN loss using a gene knockdown (KD) via siRNA in MCF7 cells. Flow cytometry and western blot analysis was utilized to investigate the molecular impact of SPEN loss. The KD and non-targeting control cells were then subjected to a high throughput kinase inhibitor screen (n=480 compounds) to identify sensitive and resistant therapeutics. Finally, we used an in-house metastatic HR+/HER2- BC patient cohort treated with CDK (cyclin-dependent kinase) 4/6 inhibitors to validate SPEN loss as a marker of resistance. Results: Using a cohort of 7519 BC samples, SPEN alterations (mutation and copy number deletions) were found to be significantly enriched in HR+ mBC vs primary HR+ disease (29% vs 7%, respectively p&amp;lt; 0.0001). SPEN altered compared with SPEN wild type (WT) HR+ mBCs were significantly associated with higher tumor mutational burden (median 4.6 vs 1.7, p &amp;lt; 0.0001), more large-scale transitions (median 20 vs 14, p= 0.006), increased fraction of genome altered (52% vs 35%, p&amp;lt; 0.0001), and enrichment of APOBEC-induced mutations (65% vs 42%, p=0.004) respectively. Taken together, these results suggest greater genomic instability in patients with tumors with SPEN loss compared with WT. This hypothesis was further supported when SPEN KD cells showed significantly increased growth rate (p= 0.04), and significantly greater DNA damage by γH2Ax staining (p=0.03) compared with control cells. In a kinase inhibitor compound screen, SPEN KD cells displayed significant resistance to the CDK4/6 inhibitor palbociclib (p&amp;lt; 0.0001) compared with WT cells. We validated this finding in vitro by demonstrating SPEN KD cell resistance to other CDK4/6 inhibitors ribociclib and abemaciclib (p= 0.02 and 0.0004 respectively). In our in-house cohort of 56 patients with HR+ mBC treated with CDK4/6 inhibitors, we found that the HR+ mBC patients with a SPEN alteration have significantly decreased overall survival (OS) compared with WT patients, (median OS 34 months vs not reached, respectively; HR 3.18, 95%CI 0.94-10.73, p=0.049). Conclusion: These results provide the first clinical evidence that SPEN alterations are enriched in HR+ mBC. Additionally, SPEN may be a biomarker for CDK4/6 inhibitor resistance in this subtype. These results warrant further analysis into the role of SPEN in BC and its relevance in clinical management. Reference: Savas P, Teo ZL, Lefevre C, et al. The subclonal architecture of metastatic breast cancer: Results from a prospective community-based rapid autopsy program "cascade". PLoS medicine 2016;13:e1002204. Citation Format: Courtney T. van Geelen, Zhi Ling Teo, Peter Savas, Stephen J. Luen, Kylie A. Clarke, Sneha Sant, Karla J. Cowley, Franco Caramia, Kaylene J. Simpson, Fabrice Andre, Sarah-Jane Dawson, Richard Pearson, Sherene Loi. SPEN is a biomarker for CDK4/6 inhibitor resistance in patients with metastatic hormone receptor positive (HR+)/HER2- breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-16.